This research program has investigated the regulatory mechanisms responsible for the expression of three nonmuscle myosin heavy chain (NMHC) genes, NMHC II-A, NMHC II-B, and NMHC II-C. We have focused on NMHC II-A gene regulation as it relates to cell type-dependent transcriptional mechanisms and NMHC II-B gene regulation as it relates to neural cell-specific alternative pre-mRNA splicing mechanisms. We have also started studying transcriptional regulation as well as tissue-dependent regulation of alternative splicing of the NMHC II-C gene. With respect to NMHC II-A gene regulation, we have been characterizing a 0.5 kb fragment in Intron 1 located 33 kb downstream from the transcriptional start sites. Sequence comparisons between the human and mouse genes revealed high levels of conservation in a region of 150 bp within the 0.5 kb fragment. Reporter gene analysis and electrophoretic mobility shift assays defined an interferon-stimulated response element as a major regulatory element. Among a large family of interferon regulatory factors, IRF-2 was found to bind this element in vitro and in intact cells. Furthermore, the results of chromatin immunoprecipitation assays and reporter gene analysis have suggested that IRF-2 is involved in activation of NMHC II-A gene transcription during differentiation of promyelocytic leukemia HL60 cells into the macrophage lineage. With respect to NMHC II-B, previous studies in this laboratory have demonstrated the existence of a neural cell-specific NMHC II-B isoform, in addition to the ubiquitously expressed form of NMHC II-B. This neural cell-specific isoform is generated by cassette-type alternative splicing of exon N30, which gives a 10 amino acid-insertion near the ATP-binding region of myosin molecules. A cis-acting intronic enhancer (IDDE), located approximately 1.5 kb downstream of N30, and an exonic enhancer located within N30, were found to be indispensable for neural cell-specific inclusion of N30. The IDDE includes two copies of the hexanucleotide motif UGCAUG. Sequence-specific RNA-binding proteins Fxh and A2BP1, which share an identical RNA recognition motif, have been demonstrated to bind this hexanucleotide motif. Fxh and A2BP1 are expressed not only in brain, but also muscles to a large extent. Interestingly, however, both mouse Fxh and A2BP1 transcripts were found to undergo tissue-specific alternative splicing, generating protein isoforms specific to brain and muscle. Co-transfection of the expression constructs for each of the isoforms with minigene reporter constructs showed that the brain isoforms of both Fxh and A2BP1 promoted N30 splicing much more efficiently than the muscle-specific isoforms. The hexanucleotide UGCAUG in the IDDE was indispensable for splicing activation by these proteins. Moreover, exogenous expression of the brain isoform of Fxh or A2BP1 resulted in an increase in N30 inclusion in the endogenous NMHC II-B mRNA. These results support the hypothesis that tissue-dependent isoforms of Fxh and A2BP1 play an important role in regulating neural cell-specific alternative splicing of NMHC II-B via the IDDE. With respect to NMHC II-C, we have identified two alternative promoters for the mouse gene, which are utilized in a tissue-dependent manner. The promoter located more distally from the common exon 2 is utilized in a wide variety of tissues including brain and heart in adult mice, whereas the more proximal promoter is predominantly utilized in lung. Both promoters lack a TATA box and show a high GC content, similar to the single promoter found for NMHC II-A and NMHC II-B. Unlike NMHC II-A and II-B, however, NMHC II-C mRNAs are expressed at the very low level at early embryonic stages as well as embryonic stem cells. Consistent with this fact, many cell lines derived from the tissues where the NMHC II-C mRNAs are expressed in adulthood express the NMHC II-C at very low levels. Using a number of cell lines, we have found that CpG methylation and histone deacetylation play critical roles in repression of the distal promoter of the NMHC II-C gene and that Sp3 plays a role in activation of this promoter.